Naturally Acquired Rabies in White-Eared Opossum, Brazil

Opossums are considered resistant to rabies. Nonhematophagous bats are reservoirs of rabies in urban areas of South America. We analyzed bats and opossums tested for rabies during 2021 in a highly urbanized city in Brazil to understand spillover in an urban setting. Wildlife surveillance is necessary to prevent rabies in humans and domestic animals.

then inoculated in murine neuroblastoma cells (4x105 cells/mL) in triplicate and the microplate was incubated at 37°C in a 5% CO2 humidified incubator for 96 hours.After the incubation period, the microplate was fixed and stained as described above and observed in a fluorescence microscope (Zeiss Axio Vert.A1) with a 20x objective in search of fluorescent infected cells.
Histopathology: Fixed samples were embedded in paraffin wax, sectioned at 4 μm-thick, and stained with hematoxylin and eosin for histologic analysis.For the CNS analysis, no degenerative changes, e.g., vacuolar degeneration and death neurons were considered due to the lack of knowledge between the time of death and the postmortem exams and collected performance.
IHC: Deparaffinized 3μm sections of tissues in silanized slides were submitted to enzymatic digestion (20' at 37°C) by proteinase K (125mg/ml).Endogenous peroxidase was blocked with 6% hydrogen peroxide (30') followed by overnight incubation with mouse hyperimmune antiserum polyclonal (in-house -Evandro Chagas Institute, Pará, Brazil) at a concentration of 1/2000.The signal was amplified by Polink-2AP Broad Kit (GBI Labs; WA; USA) for '60' conjugated to alkaline phosphatase and visualization was achieved by warp red(3′) (Polink-2 AP Broad Detection System; GBI Labs, WA, USA, cat.D24-110).The samples were counterstained with Harris Hematoxylin (20"), followed by dehydration and slide mounting with synthetic resin.Brain mammal tissue fragments known to be positive and confirmed by IHC were used as positive controls.The same steps were followed for the negative control, except for the primary antibody incubation, replaced by non-immune serum from a mouse.

RT-PCR and phylogeny:
Brain samples were submitted to RNA extraction with TRIzol (Thermo Fisher Scientific) following the manufacturer's instructions.Complementary DNA synthesis was made using the Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific) following the manufacturer's protocol.PCR was performed using Platinum Taq DNA Polymerase (Thermo Fisher Scientific), and the primers Ga3222-40 (5′ CGCTGCATTTTRTCARAGT 3′) and Gb4119-39 (5′ GGAGGGCACCATTTGGTMTC 3′) targeting the glycoprotein gene as described elsewhere (1).Gel electrophoresis was used to confirm specific product amplification.The amplicon was purified by an enzymatic method (ExoSAP-IT -Thermo Fisher Scientifc), and the BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientifc) was used for sequencing reactions.Sequences were obtained in ABI-3500.PHRED algorithm was used for base quality analysis of the sequence generated.Values equal to or higher than 20 were considered real.Next, the software BioEdit Sequence Alignment Editor v. 7.0.3(2) was used for generating the consensus sequence and multiple alignments.A database was built with sequences retrieved from GenBank representative of rabies virus lineages circulating in Brazil, and the phylogenetic reconstruction was inferred by Maximum Likelihood using PAUP 4.0 (3) with GTR+I+G as an evolutionary model with 1,000 bootstrap replicates.European Bat Lyssavirus was used as an outgroup.
Geospatial Analysis: Bats and opossums locations were georeferenced using an API in Google Sheets Geocoding service (4).The geographic coordinates were imported into ArcGIS Pro 2.9.5, of which 930 (88.6% of bats) and 18 (81.8% of opossums) locations of animal captures were successfully located.After validation, 94 bats (8.96%) and four opossums (18.18%) had to be manually geo-edited to ensure the correct locations.Each animal represented a point (shapefile) and was categorized according to its taxonomy and outcome diagnosis in the thematic maps.Spatial analysis was performed using Kernel estimator density (5).We defined a bandwidth of 1,000m to identify the density of bats by genus and overlap its information with the Opossum's location.In addition, areas of influence of 50m (buffers) were created around the location of the capture of positive animals to identify the presence of vegetation, classified as densely vegetated, remnants of vegetation, sparsely vegetated, and very sparsely vegetated, ranging from higher to lower arboreal vegetation cover, respectively (see Appendix 1 Table ).